Biomolecular receptors such as nucleic acids that switch between two or more conformations upon binding to a specific target can be used to build specific and sensitive biosensors. In this work, based on the electrochemical dual-signaling ratiometric strategy and triple-helix molecular switch, we developed a selective, reusable, and simple electrochemical DNA (E-DNA) biosensor for target DNA (T-DNA) detection. A hairpin DNA capture probe labeled with methylene blue (MB-DNA) self-assembles on the surface of a gold electrode (GE) through Au-S bond, and then a single-strand DNA modified with two ferrocenes (Fc-DNA) on each end to enhance the oxidation signal hybridizes with the MB-DNA to form a triple-helix conformation. When T-DNA exists, the Fc-DNA hybridizes with T-DNA disassembling the triple-helix stem and allowing the MB-DNA to revert to its hairpin structure. Hence, the Fc tags diffuse away from the GE surface while the MB tags remain affixed close to it, resulting in a decrease in the peak current of Fc (IFc) and an increase in that of MB (IMB). The linear relationship between the value of IMB/IFc and the T-DNA concentration is observed from 0.5 to 80 pM, and the limit of detection is as low as 0.12 pM. The developed E-DNA biosensor may have great potential in the electrochemical detection of a wide range of analytes and be a biosensing platform for early clinical diagnosis and biomedical research.